The normal mammalian immune system responds to viral infection in a variety of ways. One important response is that T lymphocytes become able to recognize and kill virus-infected cells, while leaving non-infected cells unharmed. Since viruses multiply by taking over the cell's machinery, when T lymphocytes kill the virus-infected cell they thereby limit the ability of the virus to reproduce itself.
The ability of T lymphocytes to kill only infected cells is mediated by the ability of the infected cells to produce certain "signals". These "signals" which are protein-peptide complexes called major histocompatibility (MHC) complexes, are produced by mammalian cells in response to viral infection. These complexes are then transported to the surface of the infected cells, where they are "displayed" to other cells, most notably T lymphocytes. As T lymphocytes circulate in the body, they come into contact with cells that have MHC complexes on their surfaces. If those MHC complexes have associated with them viral or foreign antigens in the form of small fragments of viral or foreign proteins, receptors on the surfaces of the T lymphocytes become activated, and the T lymphocytes are induced to kill those cells. But when T lymphocytes come into contact with cells that do not have the viral or foreign antigens associated with the MHC complexes on their surface, the T lymphocytes do not disturb them. (Yewdell, J. W., and Bennink, J. R., Cell biology of antigen processing and presentation to major histocompatibility complex class I molecule-restricted T lymphocytes, Adv. Immunol. 52:1-123 (1992)).
There are two classes of MHC complexes, class I and class II. The production and display of MHC class I complexes is fairly well understood. Infected cells are able to degrade viral proteins to some extent, and short protein pieces, or peptides, are produced as a result. These peptides are transported from the nucleus or cytoplasm to the endoplasmic reticulum (ER) or to the Golgi apparatus; the ER and Golgi apparatus are convoluted, membranous intracellular organs involved in the post-translational processing of proteins, and in their transport to the cell surface. Once inside the ER or Golgi apparatus, the peptides bind to the MHC class I protein .alpha.-chains and .beta.-2-microglobulin, to form a trimolecular complex (Townsend, A., Ohlen, C., Bastin, J., Ljunggren, H. G., Foster, L., and Karre, K. Association of class I major histocompatibility heavy and light chains induced by viral peptides, Nature 340:443-448 (1989)). This complex is then transported to the cell surface, where it can be recognized by T lymphocyte receptors. Receptors on the surface of a particular type of T lymphocytes, known as virus-specific CD8+ T lymphocytes, specifically recognize the MHC class I complexes that are formed by the combination of MHC class I proteins and peptides derived from a particular virus, and induce the CD8+ lymphocytes to kill the cells that bear those complexes.
The presentation of MHC class I complexes and their recognition by CD8+ T lymphocytes has been also implicated in a variety of human and animal afflictions other than viral infection. Perhaps the first role identified for MHC class I complexes was their role in tissue transplant rejection, which is why they are called "Major Histocompatibility Complexes" (MHC). MHC Class I complexes appear to be of particular importance in skin graft rejection. (Zijlstra, M., Auchincloss, H., Loring, J., Chase, C., Russell, P., and Jaenisch, R., Skin graft rejection by .beta..sup.2 -microglobulin-deficient mice, J. Exp. Med. 175:885-893 (1992)). In addition, a large number of autoimmune diseases are believed to be the result of CD8+ T lymphocytes attacking cells displaying MHC class I complexes. For example, there is evidence that attack by CD8+ T lymphocytes plays a role in multiple sclerosis (see Steinman, L., Autoimmune disease Sci. Amer. 269(3):106-114), diabetes (Oldstone, M. B. A., Nerenberg, M., Southern, P., Price, J., and Lewicki, H., Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response, Cell 65:319-331 (1991)), and arthritis (Braun, W. E., HIA molecules in autoimmune diseases, Clin. Biochem. 25(3):187-191 (1992); Scarpa, R., Del Puente, A., di Girolamo, C., della Valle, G., Lubrano, E., and Oriente, P., Interplay between environmental factors, articular involvement, and HLA-B7 in patients with psoriatic arthritis, Annals of Rheumatic Dis. 51:78-79 (1992)).
Although viral infection usually results in the display and recognition of MHC complexes, there are a number of animal viruses that are able to persist in the body, despite these mechanisms in the immune system that usually detect and destroy infected cells. Some such persistent viruses produce an extended or even constant infection, while others are able to become dormant or latent for long periods and then reappear to reinfect the individual. It is now recognized that some of these viruses evade detection by producing proteins that interfere with or block the cell's ability to make or display MHC class I complexes (Gooding, L. R., Virus proteins that counteract host defenses, Cell 71:5-7 (1992)).
Different persistent viruses appear to interfere with different stages in the production and display of MHC complexes. For example, the Ela gene of adenovirus type 12 produces a protein that blocks transcription of the MHC class I genes, thus preventing the production of the MHC class I proteins themselves (Schrier, P. I., Bernards, R., Vaessen, R. T. M. J., Houweling, A., and van der Eb, A. J., Expression of class I major histocompatability antigens switched off by highly oncogenic adenovirus 12 in transformed rat cells, Nature 305:771-775 (1983)). The E3 gene of human adenovirus types 2 and 5 produces a 19 thousand dalton (KD) protein that binds to the MHC class I proteins and causes them to remain sequestered or "stuck" in the ER or Golgi apparatus (Burgert, H.-G., and Kvist, S., An adenovirus type 2 glycoprotein blocks cell surface expression of human histocomoatibility class I antigens, Cell 41:987-997 (1985)). Similarly, murine cytomegalovirus produces a protein that inhibits the transport of the completed protein-peptide complexes from the Golgi apparatus to the cell surface (del Val, M., Hengel, H., Hacker, H., Hartlaub, U., Ruppert, T., Lucin, P., and Koszinowski, U. H., Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocomoatibility complex class I molecules into the media-golgi compartment, J. Exp. Med. 176:729-738 (1992)). Using an apparently very different mechanism, myxoma virus appears to cause the MHC class I proteins to be removed from the cell surface (Boshkov, L. K., Macen, J. L., and McFadden, G., Virus-induced loss of class I MHC antigens from the surface of cells infected with myxoma virus and malignant rabbit fibroma virus, J. Immunol. 148:881-887 (1992)).
Herpes simplex virus (HSV) types 1 and 2 are persistent viruses that commonly infect humans; they cause a variety of troubling human diseases. HSV type 1 causes oral "fever blisters" (recurrent herpes labialis), and HSV type 2 causes genital herpes, which has become a major venereal disease in many parts of the world. No fully satisfactory treatment for genital herpes currently exists. In addition, although it is uncommon, HSV can also cause encephalitis, a life-threatening infection of the brain. (The Merck Manual, Holvey, Ed., 1972; Whitley, Herpes Simplex Viruses, In: Virology, 2nd Ed., Raven Press (1990)).
A most serious HSV-caused disorder is dendritic keratitis, an eye infection that produces a branched lesion of the cornea, which can in turn lead to permanent scarring and loss of vision. Ocular infections with HSV are a major cause of blindness in North America. Immune responses play a major role in causing the tissue damage that results from recurrent ocular HSV infections, and T lymphocyte-mediated responses are a prominent cause of this damage. There is evidence that the CD8+ T cell subset is very important in these destructive immune responses (Hendricks, R. L., and Tumpey, M., Contribution of virus and immune factors to herpes simplex virus type I-induced corneal pathology, Invest. Opthalmol. Vis. Sci. 31:1929-1939 (1990)).
On initial infection, HSV usually produces a generalized, acute infection, which is cleared by the body's normal immune response. However, during the acute phase, some virus particles invade sensory nerve cells, and there they are able to become latent, and survive long after the acute infection has been cleared by the immune system, even though antibodies against them are abundant in the blood. They then later become re-activated and produce local infections. These are, as might be expected, fairly rapidly cleared by the already-prepared immune system. (Zweerink, H. J., and Stanton, L. W., Immune response to herpes simplex virus infections: virus-specific antibodies in sera from patients with recurrent facial infections, Infect. Immun. 31:624-630 (1981)). This cycle is quite familiar to those who are prone to "fever blisters", which appear to be caused by sunlight-induced activation of latent HSV particles in the lips.
Like certain other persistent viruses, it appears that HSV inhibits immune recognition of infected cells by interfering with the synthesis, transport or display of MHC class I complexes. One reason that this was not immediately appreciated by immunologists studying anti-HSV immunity is that in mouse models of HSV infection, the infected cells are primarily killed by HSV-specific CD8+ T lymphocytes, which specifically recognize MHC class I protein-HSV peptide complexes; this suggests that in these models, CD8+ T lymphocyte recognition is not strongly inhibited. However, in humans, the HSV-infected cells are more often specifically killed by HSV-specific T lymphocytes of another class, called CD4+, which recognize complexes composed of HSV-derived peptides and MHC class II proteins. (Schmid, D. S. and Rouse, B. T., The role of T cell immunity in control of herpes simplex virus, In: Herpes Simplex Virus: Pathogenesis, Immunobiology, and Control, B. T. Rouse, ed. (Berlin:Springer-Verlag) pp. 57-74 (1992). Furthermore, it has been found that human fibroblasts that are infected with HSV are not recognized and killed by HSV-specific CD8+ lymphocytes, but are killed by non-specific natural killer (NK) cells, which are not dependent on MHC class I complexes for recognition (Posavad, C. M. and Rosenthal, K. L., Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity, J. Virol. 66:6264-6272 (1992)). These findings suggest that recognition by CD8+ T lymphocytes is inhibited in human HSV infections.
Exactly what mechanism, what genes and what proteins might be involved in HSV's ability to suppress immune recognition has, until discovery of the present invention, remained unknown. HSV resistance to T lymphocyte recognition was known to occur within 2 to 3 hours of infection, id., but MHC class I expression on the surface of HSV-infected cells was not observed to be markedly reduced until 14-20 hours after infection (Carter, V. C., Jennings, S. R., Rice, P. L. and Tevethia, S. S., Mapping of a herpes simplex virus type 2-encoded function that affects the susceptibility of herpes simplex virus-infected target cells to lysis by herpes simplex virus-specific cytotoxic T lymphocytes, J. Virol. 49:766-771 (1984)). Furthermore, other cell-to-cell propagated inactivation mechanisms have also been observed (York, I., and Johnson, D. C., Direct contact with herpes simplex virus-infected cells results in inhibition of lymphokine-activated killer cells due to cell to cell spread of virus, J. Infect. Dis. 168:1127-1132 (1993)).
The genome of herpes simplex virus type 1 is encoded on a linear, double-stranded DNA of about 152 kilobases. The HSV-1 genome has been completely sequenced. See: McGeoch, D., M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott and P. Taylor, The Complete DNA sequence of the Long Unique Region in the Genome of Herpes Simplex Virus Type 1, J. Gen. Virol. 69: 1531-1574 (1988). The genome codes for about 76 proteins, many of which have been named according to when in the infectious cycle they are produced. The protein sequences for all of the HSV-1 proteins are known, having been deduced from their corresponding gene sequences. Furthermore, many years of research has resulted in the identification of the function for many of these proteins. Nevertheless, there are still a number of proteins encoded by the HSV-1 genome that have no known function.
One of the proteins whose function has remained unknown is the immediate-early protein ICP47. Various researchers have given this protein other names, including IE12, Vmw12, and IE5. The gene for this protein is known as US 12, and is also known as .alpha.47. The coding region of the US 12 gene is 264 base pairs long, which means that the ICP47 protein is 88 amino acids long. Although ICP47 is observed to migrate in gel electrophoresis as a protein of about 12,000 daltons, the molecular weight, as calculated from its amino acid sequence, is 9792 daltons (McGeoch, D. J., Dolan, A., Donald, S., and Rixon, F. J., Sequence determination and genetic content of the short unique region in the genome of herpes simplex virus type 1, J. Mol. Biol. 181:1-13 (1985)).
Various researchers have previously attempted to discern the function of ICP47, but prior to the present invention, without success. Deletion of the US 12 gene has been found to have no effect on infectivity (Mavromara-Nazos, P., Ackermann, M., and Roizman, B., Construction and properties of a viable herpes simplex virus 1 recombinant lacking coding sequences of the .alpha.47 gene, J. Virol. 60:807-812 (1986)), and the most recent reported effort to determine the function of ICP47 concluded that the US 12 gene plays "no important role in the establishment and/or reactivation from latency" (Nishiyama, Y., Kurachi, R., Daikoku, T., and Umene, K., The US9, 10, 11, and 12 genes of herpes simplex virus type 1 are of no importance for its neurovirulence and latency in mice, Virology 194:419-423 (1993)).
Herpes Simplex Virus type 1 is but one member of an extended family of viruses. HSV type 2 is a close relative; its genome is "collinear" with that of HSV type 1, with "reasonable, but not identical, matching of base pairs". (Whitley, Supra at 1845). Other members of the human herpesvirus family include cytomegalovirus, varicella-zoster virus, herpes virus 6, herpes virus 7, and Epstein-Barr virus. There are also more than 50 herpesviruses that infect more than 30 other animal species (Id.), including some that infect humans.
Herpes Simplex Virus Type 2 has a gene that corresponds to the HSV Type 1 US 12 gene. It maps in the same genomic location, and produces a protein that migrates as a 12,300 dalton protein on gel electrophoresis, which is similar to the migration of ICP47 (Marsden, H. S., Lang, J., Davison, A. J., Hope, R. G., and MacDonald, D. M., Genomic location and lack of phosohorylation of the HSV immediate-early polypeptide IE 12, J. Gen. Virol. 62:17-27 (1982)). We have compared these gene sequences, and have determined that the Herpes Simplex Virus types 1 and 2 ICP47 proteins are 45% identical at the amino acid level, and 60% homologous when one allows substitution of similar amino acids.
Varicella-Zoster Virus does not appear to have a gene corresponding to US 12 (Davison, A. J., and D. J. McGeoch, Evolutionary Comparisons of the S Segments in the Genomes of Herpes Simplex Virus Type 1 and Varicella-Zoster Virus, J. Gen. Virol. 67:597-611 (1986)), and the pseudorabies virus does not appear to contain a sequence corresponding to US 12 in the region encoding genes corresponding to other "unique stretch" (US) genes (Zhang, G., and D. P. Leader, The Structure of the Pseudorabies Virus Genome at the End of the Inverted Repeat Sequences Proximal to the Junction with the Short Unique Region, J. Gen. Virol. 71:2433-2441 (1990)). However, it is unclear whether the many other herpesviruses contain such genes.